Product Description
Reproducible and high-throughput in vitro models are essential for studying complex microbial communities. This kit is designed for the in vitro modeling of human mucus. Universal Bac3Gel is presented here as a platform for culturing microorganisms. The protocol describes how to culture single strains in Universal Bac3Gel using a multi-well plate format and how to prepare the samples for downstream analyses. Downstream assays include cell viability assessment, flow cytometry, metabolomics, and sequencing. The potential applications of Universal Bac3Gel include microbial mining, drug screening, and permeability assays.
Product Page: https://www.bac3gel.com/category/bac3gel
Components
2x multi-well plates containing Universal Bac3Gel.
50 mL Bac3Gel® dissolution medium (50 mM sodium citrate).
Reagent Required But Not Provided
- 0.9% (w/v) NaCl solution prepared in deionized water.
Storage Conditions
Store in a dry place inside tightly sealed containers at 2-8 °C.
This product can be stored for at least 9 months.
Precautions and Disclaimer
For R&D use only.
Procedure
Before you begin
The protocol below is provided as an example to guide researchers in the development of their own procedures. This protocol describes the specific steps for culturing single strains. However, this protocol has also been used for culturing co-cultures and microbial consortia. The Universal Bac3Gel used in this protocol can produced in the culture medium of the researcher’s choice and is composed of 50 mg.mL-1 porcine stomach type III mucin. Universal Bac3Gel’s composition is fully tunable; customized compositions are commercially available (i.e. incorporation of other mucin types, proteins, lipids and media). The cross-linking and oxygen gradients within Universal Bac3Gel are intrinsic features established during the production process. These are dynamically affected by microbial culturing.
Ensure all instruments and consumables (pipettes, falcons, and microcentrifuge tubes) are sterilized and free of contaminants.
All manipulations should be performed under aseptic conditions.
Prepare a 0.9% (w/v) sterile NaCl solution.
A. Preparation of Microbial Suspensions
1. Grow cultures of the species of interest (e.g. Escherichia coli) to the logarithmic (log) growth phase in the appropriate medium.
Centrifuge the culures to pellet the cells, then discard the supernatant.
Resuspend the cell pellet in 0.9% (w/v) NaCl solution or an appropriate culture medium.
Measure the optical density at 600 nm (OD600) of the microbial suspension.
Adjust the cell concentration as needed.
B. Culturing in Universal Bac3Gel
1. Prepare Universal Bac3Gel multi-well plates at room temperature. For this protocol, 24-well plates are used as example.
2. Add the microbial culture to each well in a 1:1 volume ratio with Universal Bac3Gel.
Example: For a 24-well plate, add 700 μL of microbial culture as each well contains 700 μL of Universal Bac3Gel.
Volumes for other plate formats:
- 6-well: 3,700 μL
- 12-well: 1,600 μL
- 48-well: 350 μL
- 96-well: 100 μL
3. Cover the plates and incubate at 37 °C (or other appropriate incubation temperature) under aerobic or anaerobic conditions for up to 72 hours depending on experimental design.
The recommended incubation period is 72 hours but it may be adjusted as required by the user.
To test the effects of molecules (e.g. biotics, active principles) on microbial cells, it is recommended to let cells stabilize in Universal Bac3Gel for 24 hours at 37 °C before adding the molecule of interest.
The multi-well plates can be sealed with a breathable sealing membrane prior to incubation to prevent cross-contamination.
The microbial cultures should be added gently (to avoid cross-contamination) to the top of Universal Bac3Gel. Do not mix.
While not a strict requirement, ensuring anaerobic conditions prior culturing in Universal Bac3Gel will prevent the loss of obligate anaerobes.
C. Dissolution of Universal Bac3Gel
1. For each well, remove the supernatant from the surface of Universal Bac3Gel by gentle aspiration, without disturbing the gel.
Wash the gels by adding 700 μL (for 24-well plates) of 0.9% (w/v) NaCl solution.
Gently aspirate and discard the supernatant.
The washing step ensures that we only consider microbes colonizing Universal Bac3Gel.
2. Add Bac3Gel® dissolution medium (50 mM sodium citrate solution) to each well. Mix thoroughly to fully dissolve Universal Bac3Gel structure.
- To calculate the volume of Bac3Gel® dissolution medium to add to each well, enter the volume (μL) of Universal Bac3Gel according to the multi-well plate typology and click Calculate:
- Transfer the homogenized samples to microcentrifuge tubes.
Full dissolution of Universal Bac3Gel can take some time depending on the plate format and on the presence of exopolysaccharides.
For sequencing: Centrifuge the samples (14,000 x g, 10 minutes), then use the pellet for DNA extraction.
For metabolomics: Centrifuge the samples (14,000 x g, 10 minutes), then collect and lyophilize the supernatant.
Other applications include assessing cell viability, performing flow cytometry analysis, measuring metabolic activity, and conducting standard microbiological characterization assays.
Troubleshooting
Problem 1
The gels are not dissolving properly or take too long to dissolve.
Potential solution
Solution 1: Increase the volume of 50 mM sodium citrate and mix.